Vascular diseases are the major cause of morbidity and mortality in the United States. Despite decades of intensive research, the mechanisms responsible for these diseases are poorly understood.
The low incidence of vascular diseases in pre-menopausal women and the rapid increase in vascular diseases, including cerebrovascular and ischemic heart disease, in women following menopause are well recognized. It is now recognized that the hormone estrogen plays a significant role in preventing atherosclerotic vascular disease. Most attempts to explain the effects of estrogen on the development of vascular disease in women focus on indirect effects of estrogen on known risk factors, such as lipid and/or carbohydrate metabolism, counterbalancing of androgen-mediated effects, or indirect effects of sex hormones on the thrombotic milieu.
Estrogen replacement therapy has been rather successful in reducing the incidence of vascular disease in post-menopausal women. It can reduce the incidence of coronary artery disease by as much as 30-50 percent. However, estrogen replacement therapy has a number of significant drawbacks, including an increased risk of endometrial cancer, an increased risk of breast cancer in women, and side effects such as endometrial bleeding and breast tenderness. In addition, though estrogen therapy may theoretically have beneficial effects for vascular disease in men as well as women, attempts to examine this possibility have been limited by adverse effects observed with currently available agents and the possibility of feminization that such therapy harbors.
There is a need for more selective therapies which have some or all of the vasoprotective benefits of estrogen therapy, but have fewer undesirable side-effects.
The present invention relates to screening methods which can be used to identify agents which inhibit vascular smooth muscle cell activation and/or proliferation or enhance vascular endothelial cell activation and/or proliferation or activate estrogen responsive genes in vascular cells. Such agents are potentially useful for treatment or prevention of vascular disease and are referred to as xe2x80x9cvasoprotective agentsxe2x80x9d or xe2x80x9canti-hypertensive agents.xe2x80x9d Preferred vasoprotective agents are relatively vasospecific, i.e., their effect on one or more types of vascular cells is more pronounced than their effect on other cell types. Treatment with such vasospecific agents will generally be associated with fewer undesirable side-effects than treatment with estrogen. Vascular disease can result from overproliferation of vascular smooth muscle cells. This proliferation can be inhibited by activation and/or proliferation of vascular endothelial cells. For example, a preferred agent would inhibit the proliferation of vascular smooth muscle cells and/or increase proliferation of vascular endothelial cells associated with the development of atherosclerosis, but not have any significant effect on cells of the reproductive system, e.g., breast cells or uterine cells.
The methods of the invention permit identification of agents which inhibit vascular smooth muscle cell proliferation directly by altering the proliferation rate of vascular smooth muscle cells. The methods of the invention also permit identification of agents which inhibit vascular smooth muscle cell proliferation more indirectly by enhancing the proliferation or activation of vascular endothelial cells, which can, in vivo, exert an inhibitory effect on the proliferation of vascular smooth muscle cells and can also promote vascular stability through an increased rate or extent of re-endothelialization.
The term xe2x80x9ccardiovascular agentxe2x80x9d refers to agents which have the potential to treat, ameliorate, or prevent disorders relating to the heart and the blood vessels or circulation. Cardiovascular agents include, for example, vasoprotective agents, antihypertensive agents, cardiomyopathy therapeutic agents, coronary heart disease therapeutic agents, and heart failure therapeutic agents.
The term xe2x80x9cvasoprotective agentxe2x80x9d refers to agents which have the potential to reduce vascular disease in patients because they inhibit, directly or indirectly, unwanted proliferation of vascular smooth muscle cells and/or enhance, directly or indirectly, endothelial cell growth or regrowth at sites of vascular injury.
The term xe2x80x9cantihypertensive agentxe2x80x9d refers to agents which have the potential to reduce blood pressure. Such agents may be useful for treating disorders which include, but are not limited to, primary hypertension, malignant hypertension, pulmonary hypertension, postpartum hypertension, medication-related hypertension, migraine, renal vascular disease, aldosterone-secreting tumor, renal disease, pheochromocytoma, post-menopausal hot flashes and flushing, Cushing""s disease, toxemia of pregnancy, or any other condition associated with alterations in blood pressure.
The methods of the invention are screening assays in which candidate agents are examined to identify vasoprotective or antihypertensive agents. One type of screening assay involves examining the effect of a candidate agent on cell proliferation and/or cell activation. Another type of screening assay involves examining the effect of a candidate agent on the expression of a gene which is responsive to estrogen (xe2x80x9can estrogen responsive genexe2x80x9d). Both screening assays involve the use of vascular cells and non-vascular cells. The use of both cell types is important because the cellular milieu is very likely to influence the effect of a candidate agent on cell proliferation, cell activation, and the expression of an given estrogen receptor responsive gene.
The term xe2x80x9cestrogen responsive genexe2x80x9d refers to a gene whose expression can be affected by the presence of an estrogen.
The methods of the invention can be used to identify agents which exert their effect through a known estrogen receptor, e.g., estrogen receptor xcex1 or estrogen receptor xcex2, or through an as yet unidentified estrogen receptor (xe2x80x9cestrogen receptor-dependent agentxe2x80x9d). Moreover, the methods of the invention can be used to identify agents which exert their effect through a receptor other than an estrogen receptor. These other receptors may recognize the same DNA binding site (recognition element) as an estrogen receptor or may bind to a recognition element which is distinct from an estrogen receptor recognition element.
The term xe2x80x9cestrogen receptorxe2x80x9d means a protein which specifically binds an estrogen (e.g., 17xcex2-estradiol or E2). Estrogen binding can be measured using any standard estrogen binding assay, e.g., the assay described by Gibson et al. (Endocrinology, 29:2000, 1991). The term encompasses, but is not limited to, both the well-known estrogen receptor xcex1 (Green et al., Nature 320:134,1986) and the more recently described estrogen receptor xcex2 (Kuiper et al., Proc. Nat""l Acad. Sci. USA 93:5925, 1996; Genbank Accession Number U57439). Estrogen receptory xcex3 is also considered to be encompassed by the claims of this invention.
The term xe2x80x9cestrogen receptor-dependent agentxe2x80x9d refers to an agent the effect of which on a cell depends on the presence of an estrogen receptor (e.g., estrogen receptor xcex1 or estrogen receptor xcex2 or some other estrogen receptor) in the cell. Accordingly, the term includes agents which have one effect on a cell when an estrogen receptor is expressed by the cell and a different effect or no effect on the same cell when an estrogen receptor is not expressed by the cell. The agent may, of course, exert other effects which are not mediated via the estrogen receptor. Tamoxifen, 17xcex2-estradiol, and ICI 182,780 are all examples of estrogen receptor-dependent agents.
In selecting a vasoprotective agent, among the undesirable side-effects to be avoided or minimized are activating or growth promoting effects on: (a) cancerous cells or pre-cancerous cells; (b) uterine cells or tissue; (c) breast cells or tissue; and (d) non-vascular, non-reproductive cells. Of course, it may not be possible to completely avoid all or even some of these undesirable side-effects in selecting a vasoprotective agent. A vasoprotective agent can be therapeutically beneficial even if its use is associated to some extent with some or all of these side effects.
In addition to identifying vasoprotective agents which avoid or minimize undesirable side effects, it is desirable to identify vasoprotective agents which are osteoprotective. The term xe2x80x9costeoprotective agentxe2x80x9d refers to agents which have the potential to reduce osteoporosis in patients because they increase the activation and/or proliferation of osteoclasts, which are responsible for bone formation, or because they inhibit activation and/or proliferation of osteoclasts, which are responsible for bone absorbtion.
Some of the methods which can be used to identify vasoprotective agents involve measuring cell activation and/or proliferation. Cell proliferation can be measured by measuring cell number, DNA content, or 3H-thymidine uptake. Cell activation can be monitored by measuring the expression of genes that are up regulated by cell activation. For example, to monitor activation of vascular smooth muscle cells, expression of inducible nitric oxide synthase or vascular endothelial cell growth factor can be measured. To monitor the activation of vascular endothelial cells, expression of prostacyclin synthase or endothelial nitric oxide synthase can be measured.
Other methods which can be used to identify vasoprotective agents involve measuring the expression of an estrogen responsive gene. The effect of a candidate agent on the expression of an estrogen receptor responsive gene can often be most easily measured through the use of a reporter construct in which the upstream regulatory region of an estrogen responsive gene (or an estrogen responsive element) is operably linked to a readily measurable protein, e.g., luciferase. Among the estrogen responsive genes whose upstream regulatory regions can be employed in the methods of the invention are the vitellogenin gene, the progesterone gene, the prolactin receptor gene, the nucleophosmin gene, and vascular endothelial cell growth factor gene.
The invention also features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;
(b) culturing non-vascular cells containing the non-endogenous reporter construct in the presence of the agent; and
(c) measuring the expression of the reporter gene in the vascular endothelial cells in the presence of the agent and measuring the expression of the reporter gene in the non-vascular cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular endothelial cells in the presence of the agent compared to the expression of the reporter in control vascular endothelial cells is greater than the expression of the reporter gene in the non-vascular cells in the presence of the agent compared to the expression of the reporter gene in control non-vascular cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;
(b) culturing non-vascular cells containing the non-endogenous reporter construct in the presence of the agent; and
(c) measuring the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent and measuring the expression of the reporter gene in the non-vascular cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent compared to the expression of the reporter in control vascular smooth muscle cells is less than the expression of the reporter gene in the non-vascular cells in the presence of the agent compared to the expression of the reporter gene in control non-vascular cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;
(b) culturing null cells containing the non-endogenous reporter construct in the presence of the agent, the null cells being cells which do not express an estrogen receptor; and
(c) measuring the expression of the reporter gene in the vascular endothelial cells in the presence of the agent and measuring the expression of the reporter gene in the null cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular endothelial cells in the presence of the agent compared to the expression of the reporter in control vascular endothelial cells is greater than the expression of the reporter gene in the null cells in the presence of the agent compared to the expression of the reporter gene in control null cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;
(b) culturing null cells containing the non-endogenous reporter construct in the presence of the agent, the null cells being cells which do not express an estrogen receptor; and
(c) measuring the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent and measuring the expression of the reporter gene in the null cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent compared to the expression of the reporter in control vascular smooth muscle cells is less than the expression of the reporter gene in the null cells in the presence of the agent compared to the expression of the reporter gene in control null cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene;
(b) culturing vascular smooth muscle cells containing the non-endogenous reporter construct in the presence of the agent; and
(c) measuring the expression of the reporter gene in the vascular endothelial cell in the presence of the agent and measuring the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular endothelial cells in the presence of the agent compared to the expression of the reporter in control vascular endothelial cells is greater than the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent compared to the expression of the reporter gene in control smooth muscle cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular smooth muscle cells in the presence of the agent;
(b) culturing null cells in the presence of the agent, the null cells being cells which do not express an estrogen receptor; and
(c) measuring the rate of proliferation of the vascular smooth muscle cells in the presence of the agent and measuring the rate of proliferation of the null cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the rate of proliferation of the vascular smooth muscle cells in the presence of the agent compared to the rate of proliferation of control vascular smooth muscle cells is less than the rate of proliferation of the null cells in the presence of the agent compared to control null cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells in the presence of the agent;
(b) culturing null cells in the presence of the agent, the null cells being cells which do not express an estrogen receptor; and
(c) measuring the rate of proliferation of the vascular endothelial cells in the presence of the agent and measuring the rate of proliferation of the null cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the rate of proliferation of the vascular endothelial cells in the presence of the agent compared to the rate of proliferation of control vascular endothelial cells is greater than the rate of proliferation of the null cells in the presence of the agent compared to control null cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells in the presence of the agent;
(b) culturing vascular smooth muscle cells in the presence of the agent, the null cells being cells which do not express an estrogen receptor; and
(c) measuring the rate of proliferation of the vascular endothelial cells in the presence of the agent and measuring the rate of proliferation of the vascular smooth muscle cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the rate of proliferation of the vascular endothelial cells in the presence of the agent compared to the rate of proliferation of control vascular endothelial cells is greater than the rate of proliferation of the vascular smooth muscle cells in the presence of the agent compared to control vascular smooth muscle cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising functional regulatory sequences derived from a growth promoting gene operatively linked to a reporter gene;
(b) culturing non-vascular cells containing the non-endogenous reporter construct in the presence of the agent; and
(c) measuring the expression of the reporter gene in the vascular endothelial cells in the presence of the agent and measuring the expression of the reporter gene in the non-vascular cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular endothelial cells in the presence of the agent compared to the expression of the reporter in control vascular endothelial cells is greater than the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent compared to the expression of the reporter gene in control smooth muscle cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising functional regulatory sequences derived from a growth promoting gene operatively linked to a reporter gene;
(b) culturing null cells containing the non-endogenous reporter construct in the presence of the agent, the null cells being cells which do not express an estrogen receptor; and
(c) measuring the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent and measuring the expression of the reporter gene in the non-vascular cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent compared to the expression of the reporter in control vascular smooth muscle cells is less than the expression of the reporter gene in the non-vascular cells in the presence of the agent compared to the expression of the reporter gene in control non-vascular cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence and absence of the agent, the non-endogenous reporter construct comprising functional regulatory sequences derived from a growth promoting gene operatively linked to a reporter gene;
(b) culturing vascular smooth muscle cells containing the non-endogenous reporter construct in the presence and absence of the agent; and
(c) measuring the expression of the reporter gene in the vascular endothelial cells in the presence of the agent and measuring the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular endothelial cells in the presence of the agent compared to the expression of the reporter in control vascular endothelial cells is greater than the expression of the reporter gene in the vscular smooth muscle cells in the presence of the agent compared to the expression of the reporter gene in control vascular smooth muscle cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising functional regulatory sequences derived from a growth arrest gene operatively linked to a reporter gene;
(b) culturing non-vascular cells containing the non-endogenous reporter construct in the presence of the agent; and
(c) measuring the expression of the reporter gene in the vascular endothelial cells in the presence of the agent and measuring the expression of the reporter gene in the non-vascular cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular endothelial cells in the presence of the agent compared to the expression of the reporter in control vascular endothelial cells is less than the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent compared to the expression of the reporter gene in control smooth muscle cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular smooth muscle cells containing a non-endogenous reporter construct in the presence of the agent, the non-endogenous reporter construct comprising functional regulatory sequences derived from a growth arrest gene operatively linked to a reporter gene;
(b) culturing null cells containing the non-endogenous reporter construct in the presence of the agent, the null cells being cells which do not express an estrogen receptor; and
(c) measuring the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent and measuring the expression of the reporter gene in the non-vascular cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent compared to the expression of the reporter in control vascular smooth muscle cells is greater than the expression of the reporter gene in the non-vascular cells in the presence of the agent compared to the expression of the reporter gene in control non-vascular cells.
The invention features a method for evaluating whether an agent is a vasoprotective agent, which method includes:
(a) culturing vascular endothelial cells containing a non-endogenous reporter construct in the presence and absence of the agent, the non-endogenous reporter construct comprising functional regulatory sequences derived from a growth arrest gene operatively linked to a reporter gene;
(b) culturing vascular smooth muscle cells containing the non-endogenous reporter construct in the presence and absence of the agent; and
(c) measuring the expression of the reporter gene in the vascular endothelial cells in the presence of the agent and measuring the expression of the reporter gene in the vascular smooth muscle cells in the presence of the agent;
whereby, an agent is identified as a vasoprotective agent when the expression of the reporter gene in the vascular endothelial cells in the presence of the agent compared to the expression of the reporter in control vascular endothelial cells is less than the expression of the reporter gene in the vscular smooth muscle cells in the presence of the agent compared to the expression of the reporter gene in control vascular smooth muscle cells.
In each of the assays described herein the expression a reporter gene in a cell grown in the presence of an agent is always corrected by comparison to the expression of a reporter gene in an otherwise identical cell grown in the absence of the agent.
The term xe2x80x9cestrogen responsive elementxe2x80x9d refers to a DNA sequence element which confers upon a gene to which it is operably linked responsiveness to the presence of an estrogen. The so-called consensus estrogen receptor xcex1 response element, is an example of an estrogen responsive element. Estrogen responsive elements can be found in the upstream regulatory region of estrogen reponsive genes, e.g., the human VEGF gene or the vitellogenin gene. While the entire upstream regulatory region of an estrogen responsive gene can be used as an estrogen responsive element, it is also possible to identify one or more smaller estrogen responsive elements within the upstream regulatory region. This is because an estrogen responsive element includes the smallest unit which confers estrogen responsiveness.
A xe2x80x9creporter genexe2x80x9d is a sequence which includes a mammalian promoter, a sequence encoding a detectable protein, e.g., luciferase, and, in most cases, a poly(A) signal sequence. Reporter genes are routinely used to investigate upstream regulatory regions. Generally, all or a portion of an upstream regulatory region of interest is position upstream (5xe2x80x2) of the reporter gene promoter and the entire construct is introduced into a selected cell type to study the effect of the selected upstream region on expression of the protein encoded by the reporter gene. Reporter genes are often inserted into vectors. For example, the pGL3-Promoter Vector (Promega, Madison, Wis.) is a vector which contains an SV40 promoter upsteam of a firefly luciferase gene (including a polyA signal sequence). The vector includes sequences required for replication of the vector in E. coli and mammalian cells.
A sequence element is xe2x80x9coperably linkedxe2x80x9d to a reporter gene when it is inserted upstream of the reporter gene promoter such that can influence expression of the protein encoded by the gene. For example, an estrogen responsive element inserted into the polylinker found upstream of the SV40 promoter in pGL3-Promoter Vector is operably linked to the luciferase reporter gene.
In various preferred embodiments the vascular, non-vascular, or null cells are stably transfected with the non-endogenous reporter construct; the vascular, non-vascular, or null cells are co-transfected with an estrogen receptor expression construct. In more preferred embodiments the estrogen receptor expression construct expresses estrogen receptor xcex1 and the estrogen receptor expression construct expresses estrogen receptor xcex2.
In another aspect the invention features a vascular endothelial cell containing an estrogen receptor xcex2 expression construct. In yet another aspect the invention features a vascular smooth muscle cell containing an estrogen receptor xcex2 expression construct.
In another aspect the invention features cells of cell line WB572. In preferred embodiments the WB572 cells contain a non-endogenous reporter construct comprising an estrogen responsive element operatively linked to a reporter gene, contain an estrogen receptor xcex1 expression construct, and contain an estrogen receptor xcex2 expression construct. In more preferred embodiments the estrogen receptor xcex1 expression construct is an inducible expression construct and the estrogen receptor xcex2 expression construct is an inducible expression construct.
In all assays entailing the measurement and comparison of cell proliferation rates the measured rate of proliferation of a given cell type in the presence of an agent is always corrected for the rate of proliferation of a control cell. The control cell is an otherwise identical cell grown the absence of the agent being tested.
When measuring and comparing proliferation rates it should be understood that some vasoprotective agents may actually reduce the rate of proliferation of certain cell types. For example, a desirable vasoprotective agent might not substantially change the rate of proliferation of a selected strain of vascular endothelial cell while decreasing the rate of proliferation of a selected strain of vascular smooth muscle cells.
In various preferred embodiments the vascular, non-vascular, or null cells are stably transfected with the non-endogenous reporter construct; the vascular, non-vascular, or null cells are co-transfected with an estrogen receptor expression construct. In more preferred embodiments the estrogen receptor expression construct expresses estrogen receptor xcex1 and the estrogen receptor expression construct expresses estrogen receptor xcex2.
In another aspect, the invention provides a method for evaluating whether an agent is a cardiovascular agent, by:
(a) culturing vascular endothelial cells containing a nitric oxide synthase (NOS) gene in the presence and absence of a predetermined amount of the agent; and
(b) measuring NOS activity in the vascular endothelial cells in the presence and absence of the predetermined amount of the agent;
whereby an agent is identified as a cardiovascular agent when the NOS activity in the vascular endothelial cells in the presence of the predetermined amount of the agent is greater than the NOS activity in the vascular endothelial cells in the absence of the predetermined amount of the agent.
In another aspect, the invention provides a method for evaluating whether an agent is a cardiovascular agent, by:
(a) culturing vascular smooth muscle cells containing a nitric oxide synthase (NOS) gene in the presence and absence of a predetermined amount of the agent; and
(b) measuring NOS activity in the vascular smooth muscle cells in the presence and absence of the predetermined amount of the agent;
whereby an agent is identified as a cardiovascular agent when the NOS activity in the vascular smooth muscle cells in the presence of the predetermined amount of the agent is greater than the NOS activity in the vascular smooth muscle cells in the absence of the predetermined amount of the agent.
In a preferred embodiment of the above aspects, the nitric oxide synthase activity may be measured within about 15 minutes after administration of the predetermined amount of the agent. In other preferred embodiments of the above aspects the cells may express endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and/or neuronal nitric oxide synthase (nNOS). In another preferred embodiment of the above aspects, the nitric oxide synthase activity may be measured by measuring production of nitric oxide. In another preferred embodiment of the above aspects, the cells may be co-transfected with an estrogen receptor expression construct. The estrogen receptor expression construct may express estrogen receptor xcex1 and/or estrogen receptor xcex2.
In another aspect, the invention provides a method for evaluating whether an agent is a cardiovascular agent, by:
(a) culturing myocytes containing a non-endogenous reporter construct in the presence and absence of a predetermined amount of the agent, the reporter construct comprising an estrogen responsive element operably linked to a reporter gene;
(b) culturing non-myocytic cells or non-estrogen receptor-expressing cells containing the reporter construct in the presence and absence of the predetermined amount of the agent; and
(c) measuring the expression of the reporter construct in the myocytes in the presence and absence of the predetermined amount of the agent and measuring the expression of the reporter construct in the non-myocytic cells or the non-estrogen receptor-expressing cells in the presence and absence of the predetermined amount of the agent;
whereby the agent is identified as a cardiovascular agent when the ratio of the expression of the reporter construct in the myocytes in the presence of the predetermined amount of the agent to the expression of the reporter construct in the myocytes in the absence of the predetermined amount of the agent is greater than the ratio of the expression of the reporter construct in the non-myocytic cells or the non-estrogen receptor-expressing cells in the presence of the predetermined amount of the agent to the expression of the reporter construct in the non-myocytic cells or the non-estrogen receptor-expressing cells in the absence of the predetermined amount of the agent.
In a preferred embodiment of this aspect, the reporter construct may include an estrogen responsive element of a nitric oxide synthase (NOS) gene. In another preferred embodiment of this aspect, the reporter gene may be selected from the group consisting of luciferase, green fluorescent protein, and chloramphenicol acetyl transferase. In another preferred embodiment of this aspect, the myocytes or cells may be stably transfected with the non-endogenous reporter construct.
In another aspect, the invention provides a method for evaluating whether an agent is a cardiovascular agent, by:
(a) culturing myocytes containing an estrogen responsive gene in the presence and absence of a predetermined amount of the agent;
(b) culturing non-myocytic cells or non-estrogen receptor-expressing cells containing the estrogen responsive gene in the presence and absence of the predetermined amount of the agent; and
(c) measuring the expression of the estrogen responsive gene in the myocytes in the presence and absence of the predetermined amount of the agent and measuring the expression of the estrogen responsive gene in the non-myocytic cells or the non-estrogen receptor-expressing cells in the presence and absence of the predetermined amount of the agent;
whereby the agent is identified as a cardiovascular agent when the ratio of the expression of the estrogen responsive gene in the myocytes in the presence of the predetermined amount of the agent to the expression of the estrogen responsive gene in the myocytes in the absence of the predetermined amount of the agent is greater than the ratio of the expression of the estrogen responsive gene in the non-myocytic cells or the non-estrogen receptor-expressing cells in the presence of the predetermined amount of v agent to the expression of estrogen responsive gene in the non-myocytic cells or the non-estrogen receptor-expressing cells in the absence of the predetermined amount of the agent.
In a preferred embodiment of this aspect, the estrogen responsive gene may be a nitric oxide synthase (constitutive or calcium-dependent); inducible nitric oxide synthase; endothelial nitric oxide synthase; neuronal nitric oxide synthase; calcium-independent nitric oxide synthase gene; endothelin-1; matrix metalloproteinases, such as matrix metalloproteinase 2; E-selectin; vascular-cell adhesion molecule; transforming growth factor xcex21; coagulation system proteins, such as platelet-derived growth factor, tissue factor, fibrinogen, protein S, coagulation factor VII, and coagulation factor XII; fibrinolytic system proteins such as plasminogen-activator inhibitor 1, tissue plasminogen activator, and antithrombin III (protein S); monocyte chemotactic protein; ion channel proteins, such as ISK and HK2 (cardiac potassium channels), and connexin 4; leptin; apolipoprotein II; apolipoproteins A, B, D, and E and Lp(a); angiotensin converting enzyme; angiotensin II receptor, type; vitellogenin; progesterone; prolactin receptor; nucleophosmin; insulin-like growth factor; insulin-like growth factor receptor; transforming growth factor xcex1; c-myc; c-fos; c-jun; INT-2 protooncogene; prostacyclin synthase; prostaglandin cyclooxygenase; prostaglandin synthase; collagen; elastin; c-fos; progesterone receptor; vascular endothelial growth factor; epidermal growth factor receptor; interleukin-6; neu; egr-1; estrogen receptor; heat shock protein 27; vascular adhesion molecules; vascular smooth muscle cell calcium channels; ryanodine receptor; FLT4 receptor tyrosine kinase; and fibroblast growth factor receptor.
In another preferred embodiment of this aspect, the estrogen responsive gene may be an endogenous, genomic gene. In another preferred embodiment of this aspect, the estrogen responsive gene is a non-endogenous gene. The estrogen responsive gene may be a nitric oxide synthase (NOS) gene, for example, an inducible nitric oxide synthase (iNOS) gene, an endothelial nitric oxide synthase (eNOS) gene, a neuronal nitric oxide synthase (nNOS) gene. In another preferred embodiment of this aspect the nitric oxide synthase gene may be a human nitric oxide synthase gene.
In other preferred embodiments of the above aspects, the myocytes or the cells may be co-transfected with an estrogen receptor expression construct expressing, for example, estrogen receptor xcex1 or estrogen receptor xcex2.
In yet other preferred embodiments, any of the assays described herein may be performed in combination with any other assay. For example, assays for evaluating whether an agent is a cardiovascular agent by determining NOS activity may be performed in combination with measuring the expression of an estrogen responsive gene or a reporter gene expressed from a non-endogenous reporter construct.
Described below are various methods for screening candidate agents to identify cardiovascular agents, for example, vasoprotective agents, antihypertensive agents, cardiomyopathy therapeutic agents, coronary heart disease therapeutic agents, and heart failure therapeutic agents.
The methods of the invention rely on examining the effect of various agents based on either its effect on the growth and/or activation of particular cell types (e.g., vascular and non-vascular) or its effect on the expression of an estrogen responsive gene in particular cell types. The cellular context is critical because the expression of a gene under the control of an estrogen receptor may also be affected by one or more additional factors, such as co-activators (e.g., SRC-1), repressors (e.g., NCOR/SMRT), or other expression modulating proteins which may be cell specific. Cell specific factors include those factors which may or may not be expressed in a given cell type or which may be expressed at different levels in different cell types. Although these additional factors can contribute to the cell-type specific nature of gene activation by a steroid hormone receptor such as the estrogen receptor, the methods of the invention do not require an understanding of these additional factors.
A cardiovascular agent can be identified based on either its effect on the growth and/or activation of particular cell types (e.g., vascular and non-vascular) or its effect on the expression of an estrogen responsive gene in particular cell types (e.g., vascular and non-vascular).
The effect of a given agent on cell growth and/or activation can be measured either directly or indirectly. Direct measurement of cell growth and/or activation involves monitoring cell proliferation or activation. Indirect measurement of cell growth and/or activation involves monitoring the expression of genes encoding cell growth promoting factors and/or monitoring the expression of genes encoding cell growth arrest factors. In general, the expression of these genes is monitored indirectly by monitoring the expression of a reporter construct in which all or part of the upstream regulatory region of the gene of interest is operably linked to a sequence encoding a readily detectable protein.
As noted above, a vasoprotective or antihypertensive agent can also be identified based on its effect on the expression of an estrogen responsive gene in particular cell types. An estrogen responsive gene can be a naturally-occurring gene which is responsive to estrogen, e.g., the vascular endothelial cell growth factor gene. The effect of an agent on the expression of such a naturally-occurring estrogen-responsive gene can be measured directly or indirectly. Direct measurement involves monitoring the expression of the estrogen responsive gene itself. One can measure the expression of a genomic copy of the gene or one can measure the expression of a non-endogenous copy of the gene, e.g., a copy introduced by transfection. Generally, expression of the estrogen responsive gene is monitored by measuring mRNA production. Alternatively, protein production can be measured. In many cases it is preferable to indirectly monitor the expression of an estrogen responsive gene by monitoring the expression of a reporter construct in which all or part of the upstream regulatory region of the estrogen responsive gene of interest is operably linked to a sequence encoding a readily detectable protein. Another type of estrogen responsive gene is a wholly synthetic gene in which sequences known to confer estrogen responsiveness are operably linked to a sequence encoding a readily detectable protein. A luciferase encoding reporter construct in which luciferase expression is under the control of a consensus estrogen receptor xcex1 recognition element is an example of such an estrogen responsive gene.
Cells
As discussed above, the methods of the invention identify an agent as a candidate cardiovascular agent by examining the effect of the agent on cell growth and/or activation or gene expression in two different cellular contexts: vascular and non-vascular. Accordingly, the methods of the invention can employ a variety of cell types.
Vascular cell lines of particular interest are: WB572 cells (spontaneously transformed human saphenous vein smooth muscle cells) and SV-E6 cells (human saphenous vein cells stably transfected with the E6 viral oncogene).
Other vascular cell lines useful in the method of the invention include, but are not limited to: A7R5 cells (spontaneously transformed rat thoratic aorta smooth muscle cells; ATCC, Bethesda, Md.; ECV304 cells (human umbilical vein vascular endothelial cells; ATCC, Bethesda, Md.); GH3B6 cells (rat pituitary cells; ATCC, Bethesda, Md.); PVEC cells (rat pulmonary vein endothelial cells; J. Tissue Culture Res. 10:9, 1986); CPA47 (bovine endothelial cells; ATCC CRL 1733); CPAE cells (bovine endothelial cells; ATCC CCL 209); EJG cells (bovine endothelial cells; ATCC CRL 8659); FBHE (bovine endothelial cells; ATCC CRL 1395); HUV-EC-C cells (human endothelial cells; ATCC CRL 1730); and T/G HA-VSMC cells (human vascular smooth muscle cells; ATCC CRL 1999).
Non-vascular cell lines useful in the methods of the invention include, but are not limited to: MCF-7 cells (human breast cancer cells; ATCC, Bethesda, Md.); ZR-75-1 cells (human breast cancer; ATCC CRL-1500); UACC-893 cells (human breast cancer; ATCC CRL-1902); RL95-2 cells (human endometrial cancer; ATCC CRL-1671); KLE cells (human endometrial cancer; ATCC CRL-1622).
Cell lines which do not express estrogen receptor xcex1, e.g., COS-1 cells, Cos-7 cells, CHO cells, HEK 293 cells, and HeLa cells, are useful in some embodiments of the invention.
Bone cell lines, e.g., UMR-106 cells (rat osteogenic sarcoma; ATCC CRL-1661) and HOS (human osteosarcoma; ATCC CRL-1543), are useful for determining whether a candidate vasoprotective agent is also osteoprotective.
Myocytes, in particular, cardiomyocytes, are particularly useful cells according to the invention.
The cells used in the screens described herein may be primary cells or cell lines. The cells may also be derived from transgenic animals and may lack various genes, for example, those encoding the estrogen receptor or nitric oxide synthase proteins.
Reporter Constructs
Reporter constructs are used to indirectly monitor the effect of an agent on the proliferation and/or activation of vascular cells and to monitor the effect of an agent on the expression of an estrogen responsive gene.
Reporters Based on Estrogen Responsive Genes
A number of reporter constructs in which all or a portion of the upstream regulatory region of an estrogen receptor responsive gene or an isolated estrogen receptor recognition element (ERE) is operably linked to a sequence encoding a detectable protein are useful in the methods of the invention.
In general, any gene which is responsive to an estrogen receptor can serve as the basis for a reporter construct. For example, the following vascular genes are potentially of interest: nitric oxide synthase (constitutive or calcium-dependent); inducible nitric oxide synthase; endothelial nitric oxide synthase; neuronal nitric oxide synthase; calcium-independent nitric oxide synthase gene; endothelin-1; matrix metalloproteinases, such as matrix metalloproteinase 2; E-selectin; vascular-cell adhesion molecule; transforming growth factor xcex21; coagulation system proteins, such as platelet-derived growth factor, tissue factor, fibrinogen, protein S, coagulation factor VII, and coagulation factor XII; fibrinolytic system proteins such as plasminogen-activator inhibitor 1, tissue plasminogen activator, and antithrombin III (protein S); monocyte chemotactic protein; ion channel proteins, such as ISK and HK2 (cardiac potassium channels), and connexin 4; leptin; apolipoprotein II; apolipoproteins A, B, D, and E and Lp(a); angiotensin converting enzyme; angiotensin II receptor, type 1; vitellogenin; progesterone; prolactin receptor; nucleophosmin; insulin-like growth factor; insulin-like growth factor receptor; transforming growth factor a; c-myc; c-fos; c-jun; INT-2 protooncogene; prostacyclin synthase; prostaglandin cyclooxygenase; prostaglandin synthase; collagen; elastin; c-fos; progesterone receptor; vascular endothelial growth factor; epidermal growth factor receptor; interleukin-6; neu; egr-1; estrogen receptor; heat shock protein 27; vascular adhesion molecules; vascular smooth muscle cell calcium channels; ryanodine receptor; FLT4 receptor tyrosine kinase; and fibroblast growth factor receptor. Other potentially estrogen responsive genes are described in Mendelsohn and Karas (Current Opinion in Cardiology 9:619, 1994) and Mendelsohn and Karas (New England Journal of Medicine 340:1801, 1999).
In some cases an estrogen responsive gene will also be a growth related gene. In these cases, the change in expression indicative of a vasoprotective or antihypertensive agent may depend on the cell type. Thus, in the following list a xe2x80x9c+xe2x80x9d indicates that preferred agents increase expression of that gene (or a reporter operably linked to the upstream control region of that gene) in the indicated cell type; a xe2x80x9cxe2x88x92xe2x80x9d indicates that preferred agents decrease expression of that gene (or a reporter operably linked to the upstream control region of that gene) in the indicated cell type. In each case the format is (preferred response in vascular endothelial cells/preferred response in vascular smooth muscle cells): prostaglandin cyclooxygenase (+/+), prostaglandin synthase (+/+), nitric oxide synthase (constitutive or calcium-dependent) (+/+), collagen (xe2x88x92/xe2x88x92), elastin (xe2x88x92/xe2x88x92), c-fos (+/xe2x88x92), progesterone receptor (+/+), vascular endothelial growth factor (+/+), epidermal growth factor receptor (xe2x88x92/xe2x88x92), interleukin-6 (+/+), neu (xe2x88x92/xe2x88x92), egr-1 (xe2x88x92/xe2x88x92), estrogen receptor (+/+), heat shock protein 27 (+/xe2x88x92), vascular adhesion molecules (xe2x88x92/xe2x88x92), vascular smooth muscle cell calcium channels (xe2x88x92/xe2x88x92), ryanodine receptor (xe2x88x92/xe2x88x92), FLT4 receptor tyrosine kinase (+/xe2x88x92), fibroblast growth factor receptor (xe2x88x92/xe2x88x92), and inducible nitric oxide synthase (+/+).
All or part of the upstream regulatory region of a naturally-occurring estrogen responsive gene can be operably linked to the sequence encoding a detectable protein in order to provide a suitable reporter construct. The portion of the upstream regulatory region used must include a sequence which confers some degree of responsiveness to estrogen.
One useful reporter construct is pmVEGF-Luc. In this reporter construct, a 1.6 kB fragment of genomic DNA located upstream of the murine vascular endothelial cell growth factor (VEGF) gene has been inserted into the pGL2-Basic luciferase reporter plasmid (Promega; Madison, Wis.). The 1.6 kB fragment includes the transcription start site, 1.2 kB of sequence upstream of the start site, and 0.4 kB of sequence downstream of the start site. Useful variants of this reporter plasmid can be made by deleting a portion of the murine VEGF control region. For example, pVEGF-Luc-Apa/Kpn is identical to pVEGF-luc except that 455 bp at the 5xe2x80x2 end of the 1.6 kB fragment have been removed; pVEGF-Luc-Mlu is identical to pVEGF-luc except that 768 bp at the 5xe2x80x2 end of the 1.6 kB fragment have been removed; and pVEGF-Luc-Sma is identical to pVEGF-luc except that 455 bp at the 5xe2x80x2 end of the 1.6 kB fragment have been removed. Similar reporter constructs can be created using upstream regulatory sequences from the human VEGF gene. For example, phVEGF-Luc is a reporter construct in which 5.1 kb genomic DNA upstream of the human VEGF coding sequence inserted into the pGL2-Basic luciferase reporter plasmid. This particular construct includes 3.4 kb of sequence upstream of the VEGF transcription start site and 1.7 kb untranslated sequence downstream of the transcription start site.
Assays Based on Estrogen Responsive Reporters
Agents which activate the expression of estrogen responsive genes in vascular cells, but not in non-vascular cells are candidate vasoprotective or antihypertensive agents. Generally, reporter constructs can be used to examine the effect of a given agent on the expression of such genes. Suitable reporter constructs can be created by placing a sequence encoding a readily detectable protein, e.g., luciferase, under the control of all or part the upstream transcription control region of an estrogen responsive gene. The portion of the upstream control region used in the reporter construct must include the recognition element(s) required for response to estrogen. Such elements can be identified based on their similarity to known estrogen receptor recognition elements or by functional dissection of the upstream regulatory region.
Alternatively, a reporter gene can be placed under the control of one or more synthetic estrogen receptor response elements. Such synthetic elements can be designed based on the sequence of known estrogen receptor response elements. Both types of constructs are referred to as estrogen responsive reporters.
To set up the assay, a selected estrogen responsive reporter is introduced into a vascular cell line (a vascular endothelial cell line or a vascular smooth muscle cell line) and a non-vascular cell line. The non-vascular cell line is preferably a cell line derived from a reproductive tissue (e.g, a breast cell line or a uterine cell line). The selected estrogen responsive reporter may also be introduced into a cell line which does not express estrogen receptor xcex1 or xcex2 (null cells). Suitable null cell lines include COS cells, HeLa cells, and HEK 293 cells. While the reporter can be introduced into the cells various cell lines by transient transfection, it is preferable that the cells be stably transfected with the reporter construct. Generally, human, murine, and/or rat cells can be used in the methods of the invention. In addition, the reporter constructs can employ the upstream regulatory sequences of human, murine, or rat estrogen responsive genes. Moreover, the cell line and the gene used as the source of upstream regulatory regions for the reporter construct do not have to be of the same species. Thus, a reporter in which the upstream control region of murine VEGF gene is operably linked to sequence encoding luciferase can be used in the screening methods of the invention.
There are three preferred formats for a screening assay which is based on an estrogen responsive reporter. All three formats involve at least two different cell types which harbor the same estrogen responsive reporter. The preferred assays use either vascular cells and non-vascular cells or vascular cells and null cells or two different types of vascular cells. However, other combinations may be used. Moreover, the vascular/non-vascular format and the vascular/vascular format can employ more than one vascular cell type (e.g., a vascular smooth muscle cell line and a vascular endothelial cell line or two different vascular endothelial cell lines) and more than one non-vascular cell type (e.g., a breast cell line and a uterine cell line). The use of two or more different cell lines of the same type in a single assay format is desirable because one can identify candidate vasoprotective agents which have the same or similar effect on several cells of the same type. The vascular/null format can also employ more than one cell line of each type. In both formats one can use two or more different estrogen responsive reporter constructs.